Contrasting requirements during disease evolution identify EZH2 as a therapeutic target in AML

Epigenetic regulators, such as EZH2, are frequently mutated in cancer, and loss-of-function EZH2 mutations are common in myeloid malignancies. We have examined the importance of cellular context for Ezh2 loss during the evolution of acute myeloid leukemia (AML), where we observed stage-specific and diametrically opposite functions for Ezh2 at the early and late stages of disease. During disease maintenance, WT Ezh2 exerts an oncogenic function that may be therapeutically targeted. In contrast, Ezh2 acts as a tumor suppressor during AML induction. Transcriptional analysis explains this apparent paradox, demonstrating that loss of Ezh2 derepresses different expression programs during disease induction and maintenance. During disease induction, Ezh2 loss derepresses a subset of bivalent promoters that resolve toward gene activation, inducing a feto-oncogenic program that includes genes such as Plag1, whose overexpression phenocopies Ezh2 loss to accelerate AML induction in mouse models. Our data highlight the importance of cellular context and disease phase for the function of Ezh2 and its potential therapeutic implications.The Huntly laboratory is funded by CRUK (program C18680/ A25508), the European Research Council (grant 647685 COMAL), the Kay Kendall Leukaemia Fund, the Medical Research Council (MRC), Bloodwise, the Wellcome Trust, and the Cambridge National Institute of Health Research Biomedical Research Centre. F. Basheer is a recipient of a Wellcome Trust PhD for Clinicians award. P. Gallipoli is funded by the Wellcome Trust (109967/Z/15/Z). We acknowledge the Wellcome Trust/ MRC center grant (097922/Z/11/Z) and support from Wellcome Trust strategic award 100140. Research in the laboratory is also supported by core funding from the Wellcome Trust and MRC to the Wellcome-MRC Cambridge Stem Cell Institute. This research was supported by the Cambridge National Institute of Health Research Biomedical Research Centre Cell Phenotyping Hub

A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify additional therapeutic targets in AML, we optimize a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. We identify 492 AML-specific cell-essential genes, including several established therapeutic targets such as $\textit{DOT1L}$, $\textit{BCL2}$, and $\textit{MEN1}$, and many other genes including clinically actionable candidates. We validate selected genes using genetic and pharmacological inhibition, and chose $\textit{KAT2A}$ as a candidate for downstream study. $\textit{KAT2A}$ inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs of diverse genotypes while sparing normal hemopoietic stem-progenitor cells. Our results propose that KAT2A inhibition should be investigated as a therapeutic strategy in AML and provide a large number of genetic vulnerabilities of this leukemia that can be pursued in downstream studies.This work was funded by the Kay Kendall Leukaemia Fund (KKLF) and the Wellcome Trust (WT098051). G.S.V. is funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA) and work in his laboratory is funded by Bloodwise. C.P. is funded by a Kay Kendall Leukaemia Fund Intermediate Fellowship (KKL888)

Rational design and validation of a Tip60 histone acetyltransferase inhibitor

Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer

Heme Oxygenase-1 Accelerates Cutaneous Wound Healing in Mice

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2nd and 3rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer

Preleukemic single-cell landscapes reveal mutation-specific mechanisms and gene programs predictive of AML patient outcomes

Acute myeloid leukemia (AML) and myeloid neoplasms develop through acquisition of somatic mutations that confer mutation-specific fitness advantages to hematopoietic stem and progenitor cells. However, our understanding of mutational effects remains limited to the resolution attainable within immunophenotypically and clinically accessible bulk cell populations. To decipher heterogeneous cellular fitness to preleukemic mutational perturbations, we performed single-cell RNA sequencing of eight different mouse models with driver mutations of myeloid malignancies, generating 269,048 single-cell profiles. Our analysis infers mutation-driven perturbations in cell abundance, cellular lineage fate, cellular metabolism, and gene expression at the continuous resolution, pinpointing cell populations with transcriptional alterations associated with differentiation bias. We further develop an 11-gene scoring system (Stem11) on the basis of preleukemic transcriptional signatures that predicts AML patient outcomes. Our results demonstrate that a single-cell-resolution deep characterization of preleukemic biology has the potential to enhance our understanding of AML heterogeneity and inform more effective risk stratification strategies

Lysine demethylases KDM6A and UTY: the X and Y of histone demethylation

Histone demethylases remove transcriptional repressive marks from histones in the nucleus. KDM6A (also known as UTX) is a lysine demethylase which acts on the trimethylated lysine at position 27 in histone 3. The KDM6A gene is located on the X chromosome but escapes X inactivation even though it is not located in the pseudoautosomal region. There is a homologue of KDM6A on the Y chromosome, known as UTY. UTY was thought to have lost its demethylase activity and to represent a non-functional remnant of the ancestral KDM6A gene. However, results with knockout mice suggest that the gene is expressed and the protein performs some function within the cell. Female mice with homozygous deletion of Kdm6a do not survive, but hemizygous males are viable, attributed to the presence of the Uty gene. KDM6A is mutated in the human condition Kabuki syndrome type 2 (OMIM 300867) and in many cases of cancer. The amino acid sequence of KDM6A has been conserved across animal phyla, although it is only found on the X chromosome in eutherian mammals. In this review, we reanalyse existing data from various sources (protein sequence comparison, evolutionary genetics, transcription factor binding and gene expression analysis) to determine the function, expression and evolution of KDM6A and UTY and show that UTY has a functional role similar to KDM6A in metabolism and development

Purification of quinoa grain mass with the use of a scarifier

Celem pracy było określenie skuteczności skaryfikacji mechanicznej, jako procesu wstępnej obróbki oczyszczania nasion komosy ryżowej odmiany Faro. Skaryfikację mechaniczną prowadzono dwoma sposobami (I sposób - skaryfikacja, przesiewanie; II sposób - przesiewanie, skaryfikacja, przesiewanie) z zastosowaniem dwóch typów skaryfikatorów: bębnowego i talerzowego. Badano efektywność oczyszczania nasion w zależności od zastosowanego sposobu oczyszczania, rodzaju skaryfikatora, czasu skaryfikacji mierzonego liczbą obrotów części roboczej skaryfikatorów (odpowiednio bębna lub talerza) oraz gradacji ścierniwa. Bez względu na typ zastosowanego skaryfikatora zaobserwowano, że zastosowanie wstępnego przesiewania przed skaryfikacją pozwala na uzyskanie lepszych efektów oczyszczania nasion (około 92%) w porównaniu do metody bez wstępnego przesiewania (około 81%). Ilość wydzielonych zanieczyszczeń była zauważalnie większa w przypadku skaryfikatora talerzowego. Zastosowanie tego rodzaju skaryfikatora wpłynęło jednak niekorzystnie na stan okrywy nasiennej, co zostało wykazane za pomocą zdjęć mikroskopowych.The objective of the study was to determine the efficiency of mechanical scarification as a process of the initial purification processing of quinoa grains of Faro variety. Mechanical scarification was carried out with two methods (I method - scarification, screening; II method - screening, scarification, screening) with the use of two types of scarifiers: a drum scarifier and a plate scarifier. Efficiency of grain purification was investigated in relation to the applied method of purification, a type of a scarifier, time of scarification measured with the number of rotations of the working parts of scarifiers (respectively of a drum or a plate) or abradant gradation. Without regard to the type of the applied scarifier, it was observed that application of the initial screening through a scarifier allows obtaining better results of grains purification (approx. 92%) in comparison to the method without initial screening (approx. 81%). The amount of the selected pollutions was noticeably bigger than in case of a plate scarifier. However, the use of this type of a scarifier negatively influenced the condition of the grain cover, which was proved with the use of microscope images

Assessing content of selected bioactive compounds in bilberry preserves

Celem pracy było określenie wpływu sposobu przygotowania przetworów z borówki czernicy na zawartość wybranych związków przeciwutleniających. Materiał do badań stanowiły przetwory otrzymane z owoców zebranych w lasach województwa kujawsko-pomorskiego. Przetwory przygotowano ze świeżych oraz mrożonych owoców według domowych receptur. Badaniom poddano owoce mrożone, dżem niskosłodzony, syrop przygotowany „na ciepło”, syrop przygotowany „na zimno” oraz zupy borówkowe w dwóch wariantach. W przetworach oznaczono ekstrakt ogólny, pH, zawartość polifenoli ogółem oraz antocyjanów. Spośród badanych produktów największą zawartością polifenoli i antocyjanów charakteryzował się syrop, który nie był poddany obróbce termicznej, tzw. na zimno, odpowiednio: 1859 mg GAE/100 g i 549 mg/100 g. Dodatek soku z cytryny do zupy przed gotowaniem ograniczył degradację antocyjanów i ich zawartość (414 mg/100 g owoców) nie różniła się istotnie (p ≤ 0,05) w porównaniu z ich ilością w zupie przygotowanej z owoców mrożonych (444 mg/100 g). Wraz z wydłużaniem czasu obróbki cieplnej następowało zwiększanie degradacji badanych związków nawet do 80 % zawartości początkowej antocyjanów.The objective of the research study was to determine the effect of the method for making bilberry preserves on the content of selected antioxidant compounds. The research material consisted of the products derived from bilberry fruits harvested in the forests in the Kuyavia and Pomerania Provinces. The preserves were made from fresh and frozen fruits according to the home cooking recipes. The following was analyzed: frozen fruits, low-sugar jam, syrup made “with heat”, syrup made “without heat” and two variants of bilberry soups. In the preserves, the following parameters were determined: total soluble solids, pH, total polyphenols, and anthocyanins. Of all the products analyzed, the thermally untreated syrup, i.e. the syrup made "without heat", was characterized by the highest level of polyphenols and anthocyanins: 1.859 mg GAE/100 g and 549 mg/100 g, respectively. The addition of lemon juice to soup before cooking reduced the degradation of anthocyanins and their content (414 mg/100g) did not significantly differ (p ≤ 0.05 ) compared to the amount thereof in the soup made with frozen fruits (444 mg/100 g). As the time of thermal treatment was extended, the degradation of the compounds increased even to 80 % of the initial content of anthocyanins